IER3IP1-mutations cause microcephaly by selective inhibition of ER-Golgi transport

Mutations in the IER3IP1 (Immediate Early Response-3 Interacting Protein 1) gene can give rise to MEDS1 (Microcephaly with Simplified Gyral Pattern, Epilepsy, and Permanent Neonatal Diabetes Syndrome-1), a severe condition leading to early childhood mortality. The small endoplasmic reticulum (ER)-membrane protein IER3IP1 plays a non-essential role in ER-Golgi transport. Here, we employed secretome and cell-surface proteomics to demonstrate that the absence of IER3IP1 results in the mistrafficking of proteins crucial for neuronal development and survival, including FGFR3, UNC5B and SEMA4D. This phenomenon correlates with the distension of ER membranes and increased lysosomal activity. Notably, the trafficking of cargo receptor ERGIC53 and KDEL-receptor 2 are compromised, with the latter leading to the anomalous secretion of ER-localized chaperones. Our investigation extended to in-utero knock-down of Ier3ip1 in mouse embryo brains, revealing a morphological phenotype in newborn neurons. In summary, our findings provide insights into how the loss or mutation of a 10 kDa small ER-membrane protein can cause a fatal syndrome. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-024-05386-x.

Cells were then stained with anti-ST6GAL1 (green), anti-GM130 (red) and Hoechst  S3).Proteins significantly different in KO1 versus control cells are color-coded as shown in the legend: early/late endosome (red) and cell growth (yellow), transport vesicle (blue) and cell junction (green)-associated.
Large circles indicate modifications rescued by the re-expression of the WT protein (n = 4 independent experiments).f-i) Pathway enrichment analysis of hits (f, g) decreased  S2.MS analysis of differentially secreted proteins in IER3IP1 KO1 versus control cells.Sheet 1. List of proteins whose secretion is modified in IER3IP1 KO1 vs. control cells (Fig. 4a).Ratios between IER3IP1 KO1 and control cells are shown (increased, blue; reduced, pink; unchanged, not highlighted).

Supplementary Table
Sheet 2. List of proteins whose secretion is modified in IER3IP1 KO1 vs. control cells, and rescued by re-expression of IER3IP1 WT (i.e., expression is not significantly changed in IER3IP1 WT vs. control cells) (Fig. 4a, c).Proteins partially rescued are highlighted in light orange.Sheet 5. GSEA Reactome analysis of proteins whose secretion is decreased in IER3IP1 KO1 vs. control cells (Fig. 4a, d).Sheet 6. GSEA GO Cell Component analysis of proteins whose secretion is decreased in IER3IP1 KO1 vs. control cells (Fig. 4a, e).Sheet 7. GSEA Reactome analysis of proteins whose secretion is increased in IER3IP1 KO1 vs. control cells (Fig. 4a, f).

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red) or (h, i) increased (blue) in the cell lysates of IER3IP1 KO1 compared to control cells.j, k) Quality control of MS analyses; mean ± SD of Pearson correlations of log2 PG.Quantities measured for the surface proteome (f) and secretome (g).Supplementary Fig. S5.IER3IP1 KO1 and re-expressed IER3IP1 p.L78P increase the number of active lysosomes.a-d) Cells were (a) incubated with Magic Red substrate MR-FR2 and Hoechst 33342 dye for 60 min and directly imaged, or (b) fixed and labeled with anti-LAMP1 and Hoechst 33342, then analyzed by high-content confocal fluorescence microscopy.Max projections of Z-stacks are shown for (b).Scale bars, 10 µm.c, d) The number of objects per image was calculated using Cell Profiler and normalized to the number of nuclei.Color-coded small circles represent Sheet 5. List of proteins identified among the enriched biotinylated proteins with the keyword "Transmembrane" ( www.uniprot.org).Sheet 6. List of proteins identified among the enriched biotinylated proteins with the keyword "Signal peptide" ( www.uniprot.org).

Sheet 3 .
List of proteins whose secretion is modified in IER3IP1 WT vs. control cells.Ratios between IER3IP1 WT and control cells are shown in blue (increased) pink (decreased) or were not highlighted (unchanged).Sheet 4. List of proteins whose secretion is modified in IER3IP1 p.L78P vs. WT cells (Suppl.Fig. S5a).Proteins are highlighted in blue (increased), pink (decreased) or not highlighted (unchanged).Proteins that are differentially secreted in IER3IP1 KO1/Control and IER3IP1 p.L78P/IER3IP1 WT are shown in bold.